Cardiovascular diseases are very common in industrialized countries; their causes are complex. A number of risk factors which parallel these diseases have, nevertheless, been defined. If an individual has one or even several of these risk factors, he/she has an increased risk for cardiovascular diseases. Among these factors are: increased blood pressure; smoking (particularly cigarettes); obesity; hypercholesterolemia; male sex; diabetes; history of cardiovascular diseases in the family.
Some risks are unavoidable; others may be influenced by changes in behavior; a third group finally may be modified by drugs. Elevated levels of cholesterol in the blood belong to the third group. A distinction has to be made between two different components, which both contain cholesterol: the so-called low density lipoproteins (LDL) and the high density lipoproteins (HDL). Elevated cholesterol levels in the LDL-fraction increase the risk of cardiovascular diseases (`bad cholesterol`), while elevated cholesterol levels in the HDL-fraction decrease the risk of cardiovascular diseases (`good cholesterol`).
Besides the already mentioned HDL and LDL blood also contains chylomicrons, very low density lipoproteins (VLDL), and intermediate density lipoproteins (IDL). HDL may be subdivided into the groups HDL.sub.1, HDL.sub.2, and HDL.sub.3. The distinction of groups and subgroups is based on different physicochemical (e.g., density, composition, morphology) and functional properties.
Drugs that lower the total concentration of cholesterol in blood have been described; there are even pharmaceuticals available which specifically lower the concentration of LDL-cholesterol while simultaneously increasing HDL-cholesterol. These therapies are based on a number of different mechanisms: on one hand, resorption of lipids in the gut may be reduced; on the other hand, it is also possible to inhibit a key enzyme of cholesterol biosynthesis (hydroxymethylglutaryl-CoA-reductase; HMG-CoA-reductase), thus lowering the synthetic rate of cholesterol. However, all these drugs (Colestipol, nicotinic acid, Lovastatin etc.) have to be taken over a long time and side-reactions cannot be excluded.
HDL are complex entities, composed of lipids (e.g., phosphatidyl choline, sphingomyelin, cholesterol and cholesteryl esters) and proteins (apoproteins; e.g., apoA-I, apoA-II, apoE). The individual components are held together by non-covalent bonds. HDL particles are continuously metabolized; they fall apart and are being rebuilt from new components, which are in turn synthesized in various organs. Another possibility to influence the ratio of LDL- to HDL-cholesterol in favor of the `good cholesterol` is therefore the addition of an excess of the protein(s) present in HDL; this will cause more cholesterol to be incorporated into HDL.
Apolipoproteins have already been isolated from HDL by conventional methods and used for substitution therapy in cases of familial an-alpha-lipoproteinemia (Tangier disease).
The traditional method for isolating HDL consists of a series of ultracentrifugation steps at various densities [Brewer et al., Methods in Enzymology 128; J. P. Segrest, J. J. Albers, edts.; Academic Press, Orlando, Fla., U.S.A., 1986]. Plasma or serum was brought to a predetermined density by the addition of solid potassium bromide; the solution was then subjected to ultracentrifugation. The upper layer was removed and more potassium bromide (solid or in concentrated solution) was added to the lower phase, which was again ultracentrifuged. With this method, lipoproteins of increasing density could be isolated sequentially.
This method is, however, completely unsuitable for industrial use, since the capacity of currently available ultracentrifuges does not exceed approximately 400 mL. The tedious method described above therefore does not yield more than a few hundred mg of protein per run. As an additional disadvantage, the plasma used is unsuitable for conventional fractionation processes described below because of its high content of potassium bromide.
According to AT-B-380 167, lipoproteins from human serum may be separated and also analyzed by sequential precipitation with polyethylene glycol. Depending on the polyethylene glycol concentration and the pH-value of the medium, either the VLDL and LDL were found in the precipitate and the HDL.sub.2 and HDL.sub.3 in the supernatant, or the VLDL, LDL, and HDL.sub.2 were in the precipitate and the HDL.sub.3 in the supernatant.
Fractions prepared according to this method, precipitates as well as supernatants, are contaminated with polyethylene glycol, which is difficult to remove from the desired proteins.
According to an earlier procedure described in FR 2 343 251, lipoproteins were separated from other blood or plasma proteins by ion exchange chromatography with a cation exchanger. Under suitable conditions, the VLDL and the LDL bound to the ion exchanger, while HDL passed the column unretarded. VLDL and LDL were eluted and recovered from the column by changing the buffer solution. The HDL could also be bound and eluted by using another cation exchanger.
LDL- and HDL-apolipoproteins, which are soluble in urea, could also be separated into individual components by chromatography with anion exchangers [H. Mezdour et al., J. Chromatogr. 414, 35-45, 1987]. The FPLC (fast protein liquid chromatography) equipment, produced by Pharmacia, Uppsala, Sweden, fitted with a Mono Q column, was particularly well suited for this purpose. The separation may then be achieved within 30 minutes. In order to obtain a sufficient purity a second isolation step is, however, required.
Chromatographic procedures for the separation of proteins are very common on a laboratory scale; they are often less suited for industrial scale processing, because they are either too expensive and/or suitable for only small amounts of material. Additionally, the solutions have to be adjusted to a protein concentration of about 2% before processing; in the case of plasma proteins, the starting material has therefore to be diluted. The diluted plasma may not be processed any longer by the procedures mentioned in the next paragraph.
Human blood plasma is nowadays collected in large amounts and processed to individual fractions; some of these fractions contain the lipoproteins. The fractions may be produced by ethanol fractionation according to a procedure originally developed in the United States and known as Cohn-Oncley-method [E. J. Cohn et al., J. Am. Chem. Soc. 68, 459-475, 1946; J. L. Oncley et al., J. Am. Chem. Soc. 71, 541-550, 1949]. Plasma fractions containing lipoproteins may also be produced by a variant of this method, the Kistler-Nitschmann procedure [H. Nitschmann et al., Helv. Chim. Acta 37, 866-873, 1954; P. Kistler and H. Nitschmann, Vox Sang 7, 414-424, 1962].